Sections fro111 white pine trccs were studied by electrori lllicroscopy in a search for the organization of cell wall layers in the pit border. Depending on the developmental stage of the trachcids, or perhaps on technical imperfections, differences appeared in the pit border within the same tree species. From an electron micrograph of a mature latewood tracheicl, a diagram was reconstructed that appears to be the most representative structure for the pit border in white pine

Murmanis, Lidija

Sachs, Irving B.

English

Wood and Fiber Science (E-Journal)

STRUCTURE OF PIT BORDER I N  PINUS STROBUS L. Lidija Murmanis and Irving B. S u c h  Forest Products L a b ~ r a t o r y , ~  Forest Service, U. S. Department of Agriculture, Madison, Wisconsin 53'705 ABSTRACT Sections fro111 white pine trccs were studied by electrori lllicroscopy in a search for the organization of cell wall layers in the pit border. Depending on the developmental stage of the trachcids, or perhaps on technical imperfections, differences appeared in the pit border within the same tree species. From an electron micrograph of a mature latewood tracheicl, a diagram was reconstructed that appears to be  the most representative structure for the pit border in white pine. INTRODUCT-ION trees, collected at various times throughout Bordered pits of coniferous tracheids, as the whole growth season. Small samples a maill route for solutes, have attracted were taken from the tree and fixed im- collsiderable attention from researchers. mediately in the fixatives used in electron Many publications on their structure have micro"0"p: KMnO.4, 0 ~ 0 4 ,  glutaraldeh~de- appeared in the literature ( Frey-Wyssling, 0 ~ 0 4 ,  and formaldehyde-glutaraIdehyde- Bosshard, and Miihlethaler 1956; Wardrop OsOl (Karnolrsky 19%). After each type Lincl Davies 19,61; Jutte and Spit 1963; Liese of fixation, the tissue was dehydrated in 1965; Fengel 1966; and Harada and Cat& graded series of acetone and embedded in 1967 ) . lIowever, hardly any two descrip- araldite-epon-lDDSA-mi~ture ( according to ti()ns of the structure on the llorders in Mollenhauer 1.964). The material was sec- these pits are completely identical. tioned with a diamond knife on a Porter- will suggcst later, the reasons for that may Blum ~1ltramicrotome. Grids were exam- manifold. ined in arl RCA-EMU 3D microscope using Studying the developmental sequence of the tracheids in Pinus strobus L. (white It isassumed that after such preparation we have seen micrographs of material, the artificially induced changes of the pit borders which, at different devel- (those caused by drying, for example) oprnental stages, would fit one or another ~hould  l'(: at the minimum. In the section- of the pit border descriptions already pub- ing, it also became apparent that it is lished. Nevertheless, when \fie scanned a difficult to obtain a section which would large number of micrographs from mature l'ass through all the constituent cell wall carlywood and mature latewood tracheid layers in the b r d e r  at exactly the same pits, u7e saw in the pit borders in a majority level. Besides, the different wall layers of cases a slightly different cell wall organ- l'robably exert a different resistance to ization from those so far described. knife passage. Consequently, wall layers ~h~ purpose of the present work is to were sometimes missing, at  other times show that a variable cell wall organization present. We found that pits of mature exists within the pit border of white pine latewood tracheids section easier because tracheids. of their smaller size and their reduce'd borders, and, consequently, provide fewer I\ZATERIAL AND METHOD artifacts from that aspect. The present observations were based RESULTS AND DISCUSSION on thin sections (mainly the transverse) from bordered pits of different \Yllite pine Four of the five tracheids shown in Fig. 1 have reached a mature state; the other, in ' Maintained a t  Madison, Wis., in cooperation which the plane Passes through the tapered with the Ur~iversity of Wiscol~sin. end, is still in the process of differentiation, 7 8 LIDIJA MURMANIS AND IR\'IiVG R. SACHS FIG. 1. Cross section of five earlywood tracheids. Bordered pit-pair is located between the tapered end of onc tracheid and the more central part of the other tracheid. Collected July 13, 1967; Knmov- sky, 3 , 2 4 0 ~ .  FIG. 2. Cross scction of bordered pit-pair between two alrnost mature tracheicls. Con~pound ~riiddle lamella (CML);  initial pit border (IPB);  S1 layer ( S ,  ); Sz layer (S2) .  Collected July 13, 1967; Karnov- sky, 8 , 2 0 0 ~ .  STRUCTURE OF PIT BORDER 9 FIG. 2A. Cross section of a young, radially expanding trachcid at a time when bordered pits first appc;ir on the radial walls. Initial pit hordcr (IPB);  primary wall ( P W ) .  Collected July 13, 1967; Karnovsky, 18,000X. 10 LIDIJA I\IUR;\IAXIS AND IRVING l3. SACHS l'ic. 3. Bordered pit-pair in differentiating earlywoocl tracheids (tangential section) showing the c,ontr;tsting initial pit border ( IPB) ;  the pit nlembranc ( P M )  with no torus yct; the Si layer ( Si) ;  ~u i t l  s:)me Sr layer (S.) on its lower sicle. Collected July 23, 1964; KMnOl, 17,000X. Frc:. 4. Differentiating tracheids with differentiation progressing from right to left. The last cell at rifil~t is n carnbial cell ( C C ) .  Tangential section. Collected July 23, 1964; KMnOa, 3,060X. as judged by its thinner sccondaly wall. opinion, corresponds to the initial pit This illustrate5 the progressiorl of the sur- border. It  is also obvious that the initial face growth of cells in the direction toward pit border is surrounded by a layer of a the cell tip. The tip border of this develop- lo~ver c'lectron opacity. This layer corre- rnentally less advanccd tracheid shows a sponds to the S1 and a portion of the S2 central contrasting zone which, in our layer; in this tracheid at this developmental STRUCTURE OF PIT BORDER 11 FIG. 5. Tangential section of a mature earlywoocl tracheicl cell wall. Coinpound rnitldle lainella ( C h l L ) ;  S, 1ayc.r (S1); S2 layer ( S ? ) ;  Sa layer (S:,). Collected July 23, 1964, I(h4n04, 15,300~.  FIG. 6. Cross section of a pit-pair between thc latewootl tracheids. Compound middle lamella (CSIL);  S, layer ( S , ) ;  S 2  layer (S.); S:; layer (S:+) ;  torus ( T ) .  Collectecl h.I:ly 29, 1964; Glutaraldehyde, 11,020 x. stage, thc S2 layer has only about one-third of the thickness of the adjacent tracheid. Fig. 2 shows a higher mabmification of a bordered pit at a later developmental stage. Here the S2 deposition is close to its com- pletion. This micrograph also shows the initial pit border as a separate zone, sur- rounded now by the S1 and S p  layers. The S3 layer is wide on the lumen side of the border but quite thin on the pit chamber side. Jiltte and Spit (1963) observed a con- trasting zone in the pit border of three coniferous species (A~aucasici, Aghatis, and Picen) studied. According to their inter- pretation, the ddrk zone corresponds to the S, layer; however, we think this to be the primary wall, classified so by its distinct- nesy from the S1 layer, rather than the time of its deposition (Fengel 1966). Its higher electron opacity probably results from the lignin incrustation, which has already started in the compound middle lamella but not yet begun in the secondary wall layers ( 1IJardrop 1965 ) . When the initial pit border first becomes visible, both the primary wall and the initial pit border have a very low and a comparable electron opacity (Fig. 2A) .  From this we conclude that the initial pit border develops before the secondary wall deposition has started, although after cleposition oj the primary wall. Conse- quently, the ~nitial pit border appears as a 12 LIDIJA hfURhIANIS AND IR\'IN<; R. SACHS Frc:. 7 .  Cross section of bordered pit-pair from differentiating earlywood tracheids. Initial pit border ( Il'B ) ;  S, layer ( S, ) ;  S z  lnycr ( S:). Collectecl July 13, 1967; Karnovsky, t i , l 2 0 ~ .  I .  8 Cross section of l~ortlercd pit-pair from mature lntewoocl trncheids. Initial pit border (IPB ) ;  S, layer (S,); S.' layer (S,). Collected January 23, 1964; OsO,, 14,000X. separate layer, as also noticed by Tliardrop and Dadswell ( 1957). According to Fengel (1966), the initial pit border (Hofanlagc) up to a certain developmental stage can be seen as an "individual" layer. Later, when the Sl layer deposition begins, the S1 overlays the initial pit border and the two cannot bc separated ~Inymorc. Harada and Cdtk ( 1967) also do not consider the initial pit border as a layer distinct from the S1 layer. On the other hand, Tliardrop and Dadswell (1957) state that the initial pit border and the S1 are two distinct layers. Frey-Wyssling et al. (1956) and Jutte and Spit (1963) consider th? initial pit border to be a part of the SI layer. The contrasting zone in the pit border is cspecially cvident after the permanganate fixation (Fig. 3 ) .  The lighter wall layer around the contrasting zone is mainly the S1 layer, with a little Ss showing on the lower side. Fig. 4 is included to show that the secondary wall formation in differentiating tracheids occurs progressively; in this case from the right to the left. Fig. 5 is a higher magnification micrograph of the cell wall. It is included to confirm that the dark central layer corresponds to the compound middle lamella; it also shows all the cell wall layers characteristic of a mature trachcid. Some of our electron micrographs of bordered pits also exhibited the structural organization described by Harada and C6t6 (1967). For example, in our Fig. 6, as in work of Harada and Cat&, the initial pit STRUCTURE OF PIT BORDER 13 FIG. 9. Pit-pair of mature latewood tracheids. Con~pound nriddle lanlella (CML) ;  Si layer (SI ) ;  SI. lager (5 - ) ;  B layer (SCi);  initial pit border ( IPB);  torus ( T )  Collected Septenlber 6, 1967; Karnovsky, 6,800X. l+c. 10. Radial section of horclcrecl pit fro111 a latewood tracheid. Pit aperture (PA) ;  S z  layer (52). Collected hlarch 20, 1964; 0~01, 1 4 , 4 5 0 ~ .  14 LIUIJA MURMANIS ~ h - D  IRVING B. SACHS FIG. 11. Radial section of bordered pit from an ( S ? ) .  Collected July 23, 1964; KMn04, 10,200X. border and the Sl layer are confluent; the S2 layer appears to end at the tip of the border; the Sg layer covers the border on the lumen side and terminates at the border tip. However, it is apparent that in our Fig. 6 the plane of section is not median, and this is responsible for the structural organization in the pit border seen in this picture. IIowever, we must add that the tracheid in our Fig. 6 is a latewood tra- chcid; whereas in Fig. 7 of Harada and CBtC., it is a "representative of micrographs made from several species," and is an early- wood tracheid. Fig. 7 shows a differentiat- ing tracheid whose bordered pit is almost identical to a figure representing the bor- dered pit of a European spruce in Jutte and Spit's article. In our Fig. 7, the initial pit border cannot be distinguished from the S1 earlywood tracheid. Pit chalnbcr ( PC ) ; S:! layer layer. We also found pictures of white pine bordered pits (Fig. 8) which corre- spond to the diagram for coniferous bor- dered pits given by Wardrop and Ilavies ( 1961). Our picture does not show the S, layer, but we often find it lacking in thin sections. Fig, 9 shows bordered pits of niature latewood tracheids. They reveal the pres- ence of the same cell wall layers as do earlywood pit borders, but the layers are more distinct in the latewood tracheid. For this reason, the latewood tracheids will be used for the generalized description of the pit border, as we visualize it in the perspec- tives of our present data. As seen from Fig. 9, the center of the border contains the initial pit border, recognizable as a zone distinct from the S1 layer. The initial pit STRUCTURE OF PIT BORDER 15 hordvr is ellveloped by the S1 layer, which thus is found on the inner and outer sides of tlicl initial pit border. The S1 is overlaid by the S.,, which is thick on the lumen side but thin 011 the pit chamber side. In the S? layer the microfibrils curve around the tip of the border; a few microfibrils extend heyolld the tip of the border and overlay the S, layer on the pit chamber side. The S2 on the lumcn side is covered by St3 which, as far as we could judge from our micrographs, ends at the tip of the border. A \xT:irty layer was not seen in any of the tracheids examined, but there are always some protoplasmic constituents present, usually in the pit chamber. Neither did we find in white pine the veil in the pit aper- turc found by Jutte and Spit (1963) in the coniferous species they examined. If Jutte and Spit consider this veil to be a remnant of the plasmalemma, it \vould meail that the protoplast has been retracted from the pit chamber to the pit aperture. Normally, the protoplasmic components fill the pit chamber and the plasmalemma out- lines the p,it chamber. Hadial sections, although so useful for replicas, do not reveal much of the different mall layer organization in the border region and are considerably more difficult to intcrprct. Figs. 10 and 11 are radial sections of bordered pits at different levels through the pit chamber. Fig. 10 shows the aperture of the pit chamber, surrounded by the Sa laycr; in Fig. 11, the plane of section passes through the pit chamber at a deeper level-between the pit membrane and pit aperture. Both Figs. 10 and 11 show that in the immediate vicinity of the pit aperture, the microfibrils run in a streamline fashion. The microfibrils at the outer zone of Sa unite with the S2 layer of the rest of cell \i7all. The pit chamb'er is filled with cel- lular contents. REPRESENTATION To summarize the results, the diagram of the white pine pit border (Fig. 12), recon- structed from Fig. 9, is compared with diagrams given by Harada and CBtk (1<)67), Juttc and Spit (19631, and War- drop and Davies (1961). When we com- pare the four schemes, we see that they all differ in one way or another. It  is also interesting to remember that Jutte and Spit (1963) stated that their scheme for conif- erous bordered pit does not agree either with that given by Trendelenburg (1939) or those ~ i v e n  by Rucher (1957) and Wardrop and Davies ( 1961). Similarly, Harada and CBtti (1967) stated that their concept for bordered coniferous pits differs from those of Jutte and Spit (1963) and \Vardrop and Davies ( 1961). According to \Vardrop and Davies and IIarada and CntC., the secondary wall deposition stops at the tip of the border, the border on the pit chamber side being covered only by the initial pit border. From our observation we assume that the S1 and the S- layers of the secondary wall continue to be depositecl around the tip of the border, and thus the pit chamber is laid out by S1 and S2 layers. Our scheme, prob- ably, comes closcbst to that of Jutte aud Spit; the main exception is that in their pictures, the S, l a y ~ r  and the initial pit border allpear as one inseparable layer, and the SH layer is a coritinuous layer covering the border along the pit chamber side and along the lumen side. In Fengel's (1966) model for a coniferous bordered pit, the Sa layer ends at the tip of the border and the S3 covers the border on the lumen side and on the pit thamber side. Our electron micrographs show that the SH layer ends at the tip of the border. However, we would think that by logical sequence of the cell wall formation, the S3 layer would follow the other secondary wall layers (S1 and S2) and cover the pit border on the pit cham- ber side. Jutte's and Spit's and Fengel's electron micrographs show that such an as~umption is not unreal. Of course, our study i~ not based on the analysis of the microfibrillar orientation in each of these layers. On the other hand, it is not certain how precisely this can be done by the present methods available. In short, we, like other worlrers, found structural varia- tions in the coniferous pit border. It  ap- pears that in some cases in white pine, these variations resulted from different develop- mental stages of the tracheids used. Some- LIDIJA MLIRMAXIS AND IRVING B. SACHS I PB s1 s2 s3 C M L  D FIG. 12. Conlparison between diagram representing our concept ( A )  and those of other researchers (B,  C ,  D ) .  A, Diagram for a borclercd pit of white pine, reconstructed from the latewood tracheid of Figure 9; B, Harada and CBtir; C, Jutte and Spit; D, Wardrop and Davies. Compound middle lamella ( CML);  initial pit border ( IPB);  middle lamella ( M L ) ;  primary wall ( P W ) ;  SI layer ( S I ) ;  S2 layer (S?) ;  S:; layer (Ss). STRUCTURE 0 1 7  PIT BORDER 17 timcs, no doubt, they resulted from tech- nical imperfections, that is, the plane of section not passing through all cell wall lavers at the same level, or, when some of the layers were lacking because of the coinprc~ssion in sectioning. Besides, it was \tatc.d by Bailey and Vestal (1937) that the cu.ra~~gcmcnt of cellulose (microfibrils) in the outer ancl central layers of the sccondarv wall "varies more or less from specimen to specimen, from traclleid to trachcid, and in different parts of the same cell." This probably would explain why so~nc,timc~s the initial pit border and the S, layer of the secondary wall are seen as scp.\ratc layers, so~netimes not. Deviations from thc prevailing orientation of micro- filxils in any tracheid are especially evident in thc pitted parts of the wall as had been noted by Bailey and Vestal (1937) and oth(.r lvorkers. Thus it seems logical that, for all these reasons. variations could exist in the organization of cell wall layers in a pit border region of tracheids in conifers. REFERENCES R.\II.EB, I. \lCT., AND M. II. VESTAL. 1937. The oricntatiorl of cellulose in the secondary wall of trnchcnry cells. J. Arnold Arboretum, 18: 185-195. I'JL~~:IIER, H. 1957. Die Tertiarlamelle von Holz- fascrii ~ i n d  ihre Erscheinungsformen bei Coni- fercir. IIolzforschung, 11 : 1-16. F ~ \ ~ : E L ,  D. 1966. Enhicklung und Ultrastruck- tur der I'inaceen-Hoftiipfel. Svensk Papper- stid., 69: 232-241. FREY-WYSSLIKG, A., H. H. BOSSHARD, AND K. MUH- LETHALEI{. 1!156. Die Submikroskopische Entwicklung clcr Hoftiipfel. Planta, 47: 115- 126. IIARADA, H., AND 'W. A. C h i ,  JR. 1967. Cell wall organization in thc pit border region of softwood tracheids. Holzforschung, 21: 81- 85. JUTTE, S. M., AND B. F. SPIT. 1963. The sub- microscopic structure of bordered pits on the radial walls of tracheids in Parana pine, Kauri, and European spruce. Holzforschung, 17: 168-175. KARNOVSKY, hi. J. 1965. A formaldehyde-gluta- raldehycle fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A-138A. LIESE, W. 1965. The fine structure of bordered pits in softwoods, p. 271-388. In W. A. CdtB, Jr. [eel.], Cellular ultrastructure of woody plants. Syracuse Univ. Press. ~ZOLLENHAUER, H. H. 1964. Plastic embedding nlixturcs for use in electron microscopy. Stain Tech., 39: 111-114. THENDELENBURG, K. 1939. Das Holz als Rohstoff. J. F. Lchinanns, Verlag, Berlin. \VARDROI', A. B. 1965. Cellular differentiation in xylt:nl. In W. A. Cat&, Jr. [ed.], Cellular ultrastructure of woody plants. Syracuse Univ. Press, pp. 61--97. , AKU H. E. DADSWELL. 1957. Variations in the cell wall organization af tracheids and fibers. Ilolzforschnng, 11 : 33-41. -- , ANU G. 'vV. DAVIES. 1961. Morpholog- ical factors relating to the penetration of liquids into wood. Holzforschung, 15: 129- 141. 

Structure of Pit Border in Pinus Strobus L.

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Structure of Pit Border in Pinus Strobus L.

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